Affymetrix MDS Cytoscan HD Project Laura Ford Genetic Technologist WMRGL

Introduction What is MDS Why Microarrays in MDS Project Overview Overview of Cytoscan Assay Project Progress

Myelodysplastic syndrome (MDS) MDS patients comprise largest diagnostic group of Oncology samples received in WMRGL ~900 samples a year. Characterised by production of abnormal myeloid cells (normal maturation and development but dysplastic) Clinically result in cytopaenias (reduction in cells) Pancytopaenia Neutropaenia Thrombocytopaenia Blasts may be present (<20%) Risk of progression to AML in 20~40%

Myelodysplastic syndrome (MDS) WHO (World Health Organisation)

Aim to Demonstrate clinical utility of Affymetrix Cytoscan HD array Project Overview Funded by Affymetrix – 600 SNP-arrays over 2 years at diagnosis and follow up of MDS/?MDS patient Equipment- Affymetrix Genechip Fluidics station Affymetrix Genechip Hybridisation oven Affymetrix Genechip Scanner Affymetric CHAS software (Chromosome analysis software) Aim to Demonstrate clinical utility of Affymetrix Cytoscan HD array G-banding is time consuming and a normal karyotype in 50% of cases - In my experience G-band analysis is only abnormal in 10% of cases, looked back at my last 100 MDS cases only 10 abnormal? Celegene- in order for prognostic scoring to identify patients who will benefit from modern treatments, including Azacytidine We do also offer as charged service if clinician requests SNP array not part of the project

Project Overview And Funded by Celgene – Analysis of MDS cases to improve G-band MDS turn around times Target ~ 21 days (14 days) Improve Turn around times and prognostic scoring to identify patients who will benefit from modern treatments, including Azacytidine G-banding is time consuming and a normal karyotype in 50% of cases - In my experience G-band analysis is only abnormal in 10% of cases, looked back at my last 100 MDS cases only 10 abnormal? Celegene- in order for prognostic scoring to identify patients who will benefit from modern treatments, including Azacytidine We do also offer as charged service if clinician requests SNP array not part of the project

Project Overview SNP Array is Complementary to G-band analysis Clinicians not charged for SNP Array testing providing the patients are confirmed MDS cases and correct consent is received for access to patient clinical history Also Run non project samples requested by clinicians as a charged service G-banding is time consuming and a normal karyotype in 50% of cases - In my experience G-band analysis is only abnormal in 10% of cases, looked back at my last 100 MDS cases only 10 abnormal? Celegene- in order for prognostic scoring to identify patients who will benefit from modern treatments, including Azacytidine We do also offer as charged service if clinician requests SNP array not part of the project

Use of SNPs Allows Characterization of LOH, UPD and consanguinity SNP Genotyping allows allele-specific copy number analysis Can use for Independent confirmation of copy number changes with SNP allelic information Enhanced ability to measure mosaicism and assess array/sample quality

Could replace chromosome analysis as front line tool for MDS testing Why Microarray in MDS? Cytogenetic changes in MDS are largely copy number Arrays offer multiplex CN analysis in a single test duplications, amplifications, deletions can detect copy neutral loss of heterozygosity (CN-LOH) Increase resolution beyond G-band chromosome Potential to use Peripheral Blood instead of Bone Marrow Could replace chromosome analysis as front line tool for MDS testing

Project Roles 50% of time G-band chromosome analysis of MDS/?MDS cases (for celgene) Improve turnaround times <21 days 50% of time Set up Affymetrix Cytoscan HD Array Competent on lab protocol Hope to prove its utility and roll out its use in front line tool for diagnosis of MDS patient Patient Selection Secure patient consent (research nurses/consultants) Data entry patient follow up (confirmed MDS) Secure follow up info when not received Update KPI’s (monthly data for Affy and Celgene) FISH training- confirm/Exclude SNP Array Results Not carrying out yet

Patient Selection DNA extracted- Qiamp Quality assessed – ?MDS/MDS routinely DNA extracted Quality assessed – minimum concentration 50ng/µL 260/280 ratio 1.8>2 260/230 ratio 1.5>2.3 Follow up and Consent received Patients need to be confirmed MDS Consent required for project (access patient records)

Cytoscan HD Platform High Density Gene level coverage Covers >36000 Ref Seq Genes High Density SNP and CN probe coverage for whole genome coverage 2.6 million markers including 750,000 SNP’s 1.9 million non-polymorphic probes Enriched Gene coverage for cancer markers

Overview of CytoScan™ Assay Digestion- restriction enzyme NSP1 used to digest DNA into fragments Ligation- uses small DNA nsp1 adaptors (linkers) ligated to the DNA to be amplified and then multiple primers annealing to the DNA adaptors using a T4 DNA Ligase to replicate whole genome. PCR – is then used to amplify DNA. Purification – using magnetic beads that attach to DNA and then washing with wash buffer Fragmentation – fragmenting the DNA into shorter pieces so it will hybridize to the Array Labelling – with a chemical that acts as a molecular glue for flourescent molecules (stain) that will be washed over chip. Hybridize to Array Chip – 16- 18 hours over night. Stained and wash off anything that hasn’t Hybridized. Scanned over night

Workflow in Lab Arial regular for the body copy… Day 1  Prep for the week run (AM) Bleach FS 2. Worksheets 3. Dilutions 4. Make 2% and 4% Gels x2 Day 2  1. Run PCR gel QC check 2. Nano Drop QC after Purification 3. Frag Gel QC after fragmentation Day 3 Prepare wash/Stains Prepare Chips only after frag gel passed Upload Batch Registration Files 5. Chip loading  Loading check. 16 samples 3.5 day protocol with day 2 being 8.30am to 6pm (Affy markets it at 2.5 day protocol). Day 3.5  CEL files uploaded to be processed CEL files uploaded to CHAS 2 x Samples spreadsheets to be updated with QC results and Lot numbers.

Example: Loss on Cytoscan Arial regular for the body copy… Example of the end result on CHAS. Scanned signal pattern generates log ratio.

84 samples run on SNP array What have we done so far? 84 samples run on SNP array 43 analysed 34 reported and results issued 24 normal results found, 10 abnormal 9 cases are undergoing further investigation Cases that failed G-banding 4 cases normal by SNP array 2 cases abnormal by SNP array

Summary Results Table Case number Karyotype SNP array Karyotype and SNP match? 1 normal yes 2 3 4 5 6 7 8 9 10 11 fail no 12 13 14 15 +12 16 Idic der(20q) 17 del(5q) 18 +8 19 20 Abnormal 46,XX,del(5)(q1q3)[5]/ 46,XX,del(5)(q1q3),del(11)(q23)[2]/ 45,XX,del(5)(q1q3),der(7;11)(q10;q10)del(11)(q23)[2]/ 46,XX[1] arr 5q14.3q33.3(72,739,251-83,696,150)x1,7pterp11.2 (1-57,601,477)x1,11p11.12q13.1(50,036,272-63,240,914)x1,11q13.1q13.1(64,501,919-65,446,765)x1,11q13.4q14.1(72,699,585-79,896,194)x3,11q14.1qter (84,666,715-134,942,625)x1

Could SNP arrays replace G-banding as front line tool for MDS testing? Unanswered Questions? Are the results from SNP comparable to G banding? Is anything extra found or missed by using one technique over the other? Can we use blood instead of marrow? Can we reduce Turn around Times? Is it cost effective? Could SNP arrays replace G-banding as front line tool for MDS testing?

Sally Jeffries – Project Lead Emma Huxley – Project Manager Acknowledgements Sally Jeffries – Project Lead Emma Huxley – Project Manager Nicola Trim – Project Researcher Sue Rose and Jane Soden Thank you Extraction Hub Array Hub

Any Questions?